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Plant Physiol. (1998) 118: 1243-1252
Ozone Sensitivity in Hybrid Poplar Is Correlated with a
Lack
of Defense-Gene Activation1
Jennifer Riehl Koch,
Amy J. Scherzer,
Steven M. Eshita, and
Keith
R. Davis*
Forestry Sciences Laboratory, United States Department of
Agriculture Northeastern Forest Experiment Station, Delaware, Ohio
43015-8640 (J.R.K., A.J.S., S.M.E.); and the Department of Plant
Biology and the Plant Biotechnology Center, The Ohio State University,
Columbus, Ohio 43210-1002 (K.R.D.)
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ABSTRACT |
Ozone is a major gaseous
pollutant thought to contribute to forest decline. Although the
physiological and morphological responses of forest trees to ozone have
been well characterized, little is known about the molecular basis for
these responses. Our studies compared the response to ozone of
ozone-sensitive and ozone-tolerant clones of hybrid poplar
(Populus maximowizii × Populus
trichocarpa) at the physiological and molecular levels.
Gas-exchange analyses demonstrated clear differences between the
ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although
ozone induced a decrease in photosynthetic rate and stomatal
conductance in both clones, the magnitude of the decrease in stomatal
conductance was significantly greater in the ozone-tolerant clone.
RNA-blot analysis established that ozone-induced mRNA levels for
phenylalanine ammonia-lyase, O-methyltransferase, a
pathogenesis-related protein, and a wound-inducible gene were
significantly higher in the ozone-tolerant than in the ozone-sensitive
plants. Wound- and pathogen-induced levels of these mRNAs were also
higher in the ozone-tolerant compared with the ozone-sensitive plants.
The different physiological and molecular responses to ozone exposure
exhibited by clones 245 and 388 suggest that ozone tolerance involves
the activation of salicylic-acid- and jasmonic-acid-mediated signaling
pathways, which may be important in triggering defense responses
against oxidative stress.
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INTRODUCTION |
Ozone is believed to cause more damage to forest trees than any
other gaseous pollutant. Ambient concentrations of ozone have increased
1% to 2% per year during the past 20 years in Europe and the United
States (Stockwell et al., 1997 ) and show no indication of leveling,
particularly in developing industrialized areas (Chameides et al.,
1994). Acute stress from exposure to high concentrations of ozone, even
for short periods of time, generally results in visible injury. Chronic
ozone stress, resulting from exposure to low concentrations over a long
period of time, generally produces little or no visible injury but
results in biochemical and physiological changes that lead to reduced
vigor and growth (Heath and Taylor, 1997). Ozone has been demonstrated
to alter basic metabolic processes of trees, including reducing
photosynthetic rate (Reich and Amundson, 1985; Coleman et al., 1995b),
decreasing Rubisco quantity and activity, reducing foliar conductance
(Pell et al., 1992; Farage and Long, 1995; Paakkonen et al., 1996), and
accelerating leaf senescence (Coleman et al., 1995b). Altered patterns
of carbon allocation have also been reported, resulting in a reduction
in winter storage pools (Coleman et al., 1995a). These alterations of
critical metabolic processes can lead to an increased susceptibility to
biotic and abiotic stressors and contribute to forest decline (McLaughlin, 1985; Schmieden and Wild, 1995).
Although the physiological responses of forest trees to ozone have been
well characterized, very little is known about the responses at the
molecular level. It is believed that when ozone enters the mesophyll
through the stomata, it is rapidly degraded, generating AOS (Kanofsky
and Sima, 1995). The presence of AOS activates defense mechanisms that
may operate by preventing the formation of AOS and/or by scavenging
them once they are formed. Glutathione and superoxide dismutase,
proposed components of these oxidative defense systems, have been shown
to increase upon ozone exposure in poplar (Populus spp.)
(Sen-Gupta et al., 1991). Total cellular activities of superoxide
dismutase, guaiacol peroxidase, and glutathione reductase were shown to
increase in birch as a result of ozone exposure (Tuomainen et al.,
1996).
A variety of plant systems have exhibited ozone induction of mRNAs for
defense-related genes known to be induced by pathogens and other
stresses (for review, see Kangasjärvi et al., 1994; Sandermann,
1996; Sharma and Davis, 1997). This overlap of induced gene expression
is likely because many of these stresses produce AOS. AOS trigger many
different pathways, some of which require SA as a signal molecule.
AOS-regulated pathways include SAR and the HR (Mehdy et al., 1996; Lamb
and Dixon, 1997), which are characterized by the induction of PR
proteins. By using cross-reacting antibodies to PR proteins from
herbaceous plants, it was demonstrated that ozone caused increased PR
protein levels in Norway spruce (Kärenlampi et al., 1994). This
induction of PR proteins by ozone is also well documented in several
herbaceous plant species (Sandermann, 1996; Sharma and Davis, 1997).
The phenylpropanoid-biosynthetic pathway, which produces a number of
defense metabolites, is also induced by ozone. Transcript levels for
enzymes involved in this pathway, including
PAL in birch (Tuomainen et al., 1996) and
cinnamoyl alcohol dehydrogenase in Norway spruce (Galliano et al.,
1993), were shown to be induced by ozone. The induction of
PAL gene expression by ozone has been shown not to require
SA in Arabidopsis, suggesting that ozone activates a SA-independent
signal transduction pathway as well (Sharma et al., 1996). Further
evidence for a second signal transduction pathway in ozone responses
was recently reported by Örvar et al. (1997), who
demonstrated that mechanical wounding or the direct application of JA
(a known mediator of wound responses) before ozone exposure decreased
the amount of ozone injury in tobacco plants.
To further elucidate the molecular mechanisms of ozone responses in
tree species, we have chosen hybrid poplar (Populus
maximowizii × Populus trichocarpa) as a model
forest tree. The advantages of using hybrid poplar for molecular
studies include a small genome size, ease of vegetative propagation,
well-developed transformation and regeneration protocols, and the
availability of ozone-sensitive and ozone-tolerant clonal lines. In our
current studies, comparisons of physiological and molecular responses
to ozone were made between an ozone-sensitive and an ozone-tolerant
clone. Expression patterns of genes representing a variety of
stress-response pathways were examined, including PAL (the
first enzyme in the phenylpropanoid biosynthesis pathway),
OMT (an enzyme involved in lignin formation), PR-1 (a PR protein characteristic of SA-mediated responses,
including SAR and HR), and WIN3.7 (a wound-inducible trypsin
inhibitor; Bradshaw et al., 1989). Clear differences in the pattern of
expression of these genes in the ozone-tolerant and ozone-sensitive
plants were observed. To determine if the ozone-sensitive plants are more susceptible to other stresses, wounding and pathogen-infection experiments were performed. A similar difference in defense-gene expression was observed, suggesting an overlap not only between ozone-
and SA-mediated pathways, but also between ozone-, pathogen-, and
wound-induced pathways in hybrid poplar.
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MATERIALS AND METHODS |
Plant Growth
Initial greenwood cuttings of hybrid poplar (Populus
maximowizii × Populus trichocarpa) that had
previously been designated as ozone sensitive (clone 388) or ozone
tolerant (clone 245), based on visible lesion formation in response to
a single acute dose of ozone (Wood and Coppolino, 1972), were obtained
from E. Pell (The Pennsylvania State University, University Park).
Cuttings were placed in sand, kept under mist until roots were
established, and then transplanted into 15-cm-diameter pots containing
Metromix 500 (Hummert, St. Louis, MO), amended as described by Pell et al. (1995). A drip-irrigation system delivered approximately 300 mL of
water to each cutting daily. All side shoots were pruned so that each
plant consisted of a single stem. No pruning was performed for at least
1 week before ozone fumigation, wounding, or pathogen infection.
Ozone Fumigation
Six weeks after transplantation, cuttings were transferred to
growth chambers modified for ozone fumigation and were acclimated for 2 to 3 d before treatment. Growth-chamber conditions averaged 25°C,
67% RH, and 370 ppm CO2, with a 14-h photoperiod
averaging 200 µmol m 2
s1 at the top of the canopy. The cuttings were
then treated for 6 h per d (9 AM to 3 PM)
with ozone at 300 ± 50 ppb for 4 d or maintained in ambient
air (<30 ppb ozone). Ozone was generated with an Orec ozone generator
(model 03V10-0, Ozone Research and Equipment, Phoenix, AZ) and each
treatment was replicated for a total of four chambers. At 3, 6, 12, 24, 30, 54, and 78 h after the start of the fumigation period, the
second and fourth fully expanded leaves were collected from eight
plants per clone per treatment, frozen in liquid nitrogen, and stored
at  80°C until used for RNA isolation.
Gas-Exchange Analysis
Gas exchange was measured under controlled conditions after 2 and
4 d of fumigation by placing the fourth fully expanded leaf in a
1-L cuvette of a photosynthetic system (model 6200, Li-Cor, Lincoln,
NE). Photosynthetic rates and stomatal conductance were measured at
225 ± 25 µmol m2
s1 using a cool-beam PAR lamp (model 300 PAR
56/2 WFL, General Electric) as the light source after a 30-min
acclimation period. The Li-Cor cuvette was flushed with outside air for
several minutes, and conditions were allowed to stabilize before
measurements were taken. Cuvette conditions averaged 23°C, 350 ppm
CO2, and 45% RH. Measurements were taken between
1 and 4 PM on four plants per chamber per clone. This
experiment used a completely randomized design, and analysis of
variance, with the chamber as the experimental unit and the plants as
the subsamples, was used to determine differences in gas exchange
attributable to ozone treatment, clone, and number of days of exposure.
Statistical Analysis Software (SAS Institute, 1989) was used for all
statistical analyses, and data presented include means ± SE.
Wounding Experiments
Mechanical wounding of cuttings was performed by crimping the
leaves with pliers. Each leaf from the fourth fully expanded leaf on
down was wounded 20 times at 9 AM, and this was repeated at
11 AM, 1 PM, and 3 PM every day for
4 consecutive d. At 3, 6, 24, 36, 54, and 78 h after the
initiation of wounding, the second and fourth fully expanded leaves
were collected, frozen in liquid nitrogen, and stored at 80°C until
used for RNA isolation.
Bacterial Infection Experiments
Overnight cultures of Pseudomonas syringae pv
maculicola KD4326 (Wanner et al., 1993) were diluted in 10 mM MgCl2 to a final A600 of 0.1. The bacterial suspension was
then hand infiltrated into the undersides of the second through the
fifth fully expanded leaves using a plastic 1-mL syringe without a
needle. The infiltrated area of the leaf was outlined with a marker and
collected at 3, 6, 12, or 24 h after infiltration for RNA
analysis. Mock inoculations were performed by infiltrating with 10 mM MgCl2 alone.
Gene Probes
Clones for poplar PAL, OMT, and
WIN3.7 were kind gifts from C. Douglas (University of
British Columbia, Vancouver), (Subramaniam et al., 1993); M. Van
Montagu (University of Gent, Belgium), (Dumas et al., 1992); and M. Gordon (University of Washington, Seattle), (Bradshaw et al., 1989);
respectively. A probe for PR-1 was generated by
reverse-transcriptase PCR using total RNA isolated from ozone-treated clone 245 plants and degenerate nucleotide primers
(5 -GCNCARAAYTCNCCNCARGAYTA-3 and 5-CCANACNACYTGNGTRTARTG-3)
corresponding to conserved amino acid sequences AQNSP/QDY and HYTQVVW,
respectively. The 310-nucleotide PCR product was cloned into a
TA-vector (Invitrogen, San Diego, CA), sequenced, and compared with
other known plant PR-1 genes. This analysis demonstrated
that the PCR product had approximately 68% nucleotide identity with
other PR-1 genes.
RNA Extraction and Analysis
Total RNA was isolated as described by Parsons et al. (1989).
Fifteen micrograms of total RNA per lane was fractionated on 1.5%
formaldehyde agarose gels in Mops buffer, pH 7.0, and transferred by
capillary blotting onto a Duralon membrane (Stratagene). RNA was
cross-linked to membranes by UV irradiation and then prehybridized for
1 h at 42°C in 6× SSC, 1% SDS, 2× Denhardt's solution, 25 µg/mL denatured salmon-sperm DNA, and 50% formamide. DNA probes were
labeled by random priming (RadPrime, BRL) and hybridized at 42°C for
16 to 18 h. Filters were washed with 2× SSC containing 1% SDS at
55°C for 15 min, followed by a 30-min wash in 0.5× SSC containing
1% SDS at 65°C. Filters were then exposed to phosphor imager screens
(Molecular Dynamics, Sunnyvale, CA) and the signal intensity was
quantified using ImageQuant software (Molecular Dynamics). The data
shown have been corrected for loading differences by quantitating
counts obtained by rehybridizing with a 28S ribosomal gene probe from
pea (Wanner and Gruissem, 1991). All experiments were performed at
least twice, with two replicates per test condition. The data shown are
from representative experiments.
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RESULTS |
Lesion Development
To confirm the different ozone sensitivities of the hybrid poplar
clones 388 and 245, cuttings were exposed to 300 ppb ozone for 6 h
daily for 5 d. Visible signs of ozone damage first appeared in
both clones at 12 to 24 h after the start of the initial 6-h ozone
fumigation and peaked at 36 h, with little additional injury occurring on subsequent days of exposure (Fig.
1). The vast majority of injury was
observed on mid-aged and older leaves, with only a modest degree of
injury occurring in leaves that were not yet fully expanded. The
ozone-sensitive clone 388 plants displayed large necrotic regions on
30% to 80% of all leaves, and generally had at least one leaf that
was covered with lesions over 50% or more of its area. Ninety percent
of the clone 388 plants fumigated with ozone developed some type of
injury, including stipple and large and small necrotic lesions
(Environmental Protection Agency, 1976). The tolerant clone 245 plants
developed stipple or flecks, but did not exhibit the extensive regions
of necrosis observed in clone 388 plants. Only 30% of the clone 245 plants developed any lesions, and of those that did, 20% or less of
the leaves had visible ozone damage.
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| Figure 1.
Comparison of ozone-induced leaf injury observed
in the ozone-tolerant clone 245 (left) and the ozone-sensitive clone
388 (right). Cuttings were grown in the greenhouse for 6 to 8 weeks
before being transferred to chambers, where they were allowed to
acclimate for 2 to 3 d before treatment with 300 ppb ozone for
6 h daily. The photograph was taken 36 h after the start of
the first 6 h of ozone fumigation.
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Effects of Ozone on Photosynthesis and Gas Exchange
Before ozone treatment, 6-week-old cuttings of the two clones
exhibited different patterns of growth and physiology. Clone 245 plants
were on average 8.5 cm taller than clone 388 plants (59.5 ± 0.7 versus 51.1 ± 0.6 cm, respectively), and had about 1.5 times as
much aboveground biomass (6.54 ± 0.44 versus 4.36 ± 0.16 g, respectively) and 1.8 times as much leaf area (1386 ± 87 versus 778 ± 30 cm2, respectively).
Both clones averaged 20 leaves each, resulting in a larger average leaf
size for clone 245. Light-response curves (data not shown) indicated
that the photosynthetic capacity of clone 388 was significantly (P < 0.05) lower than that of clone 245 when measured under saturating
light. However, the two clones demonstrated similar rates of
photosynthesis when measured under low-light conditions, simulating the
conditions used during ozone exposure.
After 2 d of exposure, ozone significantly (P < 0.05)
reduced photosynthetic rates in both clones (Fig.
2). Statistical analysis indicated that
the absolute values of photosynthesis were not significantly different
between the two clones. However, the percentage reduction was greater
in clone 245 than in clone 388 (72% versus 56%). Ozone continued to
reduce photosynthetic rates throughout the study, and by the 4th d of
fumigation, all of the ozone-treated plants had photosynthetic rates
that were less than those of the ambient-air-grown plants, and many had
negative net photosynthetic rates, indicating that the leaf tissue was
respiring more than photosynthesizing (Fig. 2).
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| Figure 2.
Effects of ozone on net photosynthesis rate
(Pn) and stomatal conductance (gs).
Measurements were made on the fourth fully expanded leaves of
ozone-tolerant clone 245 plants or ozone-sensitive clone 388 plants
after 2 and 4 d of exposure to ambient air or a 6-h daily
treatment with 300 ppb ozone. Bars represent the mean of eight plants
±SE. Results of analysis of variance produced the
following P values for photosynthesis (ozone = 0.0035, clone = 0.7058, day = 0.0036, ozone × clone = 0.1477, ozone × day = 0.0081, and clone × day = 0.7137) and stomatal conductance (ozone = 0.0150, clone = 0.0906, day = 0.0001, ozone × clone = 0.0002, ozone × day = 0.0001, and clone × day = 0.0026).
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Ozone exposure reduced stomatal conductance as well, and the magnitude
of this reduction was significantly different (P < 0.05) between
the clones (Fig. 2). After 2 d of exposure to ozone, stomatal
conductance of the ozone-sensitive clone 388 was reduced by
approximately 30% compared with a reduction of 80% in the
ozone-tolerant clone 245. After 4 d of exposure, this same
reduction of 30% was present in clone 388, but the difference between
ozone-treated and ambient-air-grown plants for clone 245 was only 12%.
The change in percentage reduction from d 2 to 4 in clone 245 was
caused by a large reduction in stomatal conductance values in the
ambient-air-grown plants combined with an increase in the stomatal
conductance values in the ozone-fumigated plants. This change in
ambient-air-grown clone 245 plants also accounted for the significant
ozone-by-day and clone-by-day interactions.
Ozone-Induced Gene Expression
Because PAL induction is often a useful indicator
of a general, coordinate plant defense response, we tested whether
sensitive and tolerant clones differed in their accumulation of
PAL transcripts in response to ozone. RNA-blot hybridization
studies demonstrated ozone-induced accumulation of PAL
transcripts in both the sensitive and tolerant clones (Fig.
3A). However, this increase was 3- to 5-fold greater in clone 245 than in clone 388. An early, transient induction of PAL mRNA was observed in both clones, with the
highest levels observed at 3 h after the initiation of ozone
exposure. Levels of PAL mRNA returned to those seen in the
controls by 24 h. Trees that were treated with ozone for 4 consecutive d also showed induction of PAL mRNA each day
(Fig. 3B). Transcript accumulation for OMT, an enzyme involved in
lignin biosynthesis, was also induced by ozone. OMT mRNA
reached the highest levels 12 h after the start of ozone treatment
and, like PAL mRNA, returned to near control levels by
24 h (Fig. 3A). As was observed for PAL mRNA induction, OMT transcripts were induced each day during the 4-d
treatment period (Fig. 3B).
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| Figure 3.
Comparison of the ozone-dependent induction
of defense-related gene mRNA accumulation in the hybrid poplar clones
245 and 388. Total RNA was extracted from the combined second and
fourth fully expanded leaves of 6- to 8-week-old cuttings that were
exposed to either ambient air or 300 ppb ozone for 6 h. The amount
of hybridizing radioactivity was quantitated using a phosphor imager
and is expressed as relative counts. A, RNA accumulation during the
first 24 h after the start of the first 6-h ozone treatment. B,
RNA accumulation measured on sequential days after a 6-h ozone
treatment.
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Previous work in this laboratory has demonstrated that Arabidopsis has
ozone-inducible defense mechanisms that provide some protection, and
that the induction of this defense requires a SA-dependent signaling
pathway (Sharma et al., 1996). We used PR-1 as a marker gene
for a SA-mediated response and tested for mRNA accumulation in response
to ozone to determine if induction of this pathway differed between the
sensitive and tolerant hybrid poplar clones. In clone 388, very little
(3-fold) induction of PR-1 by ozone was observed (Fig. 3A).
However, the tolerant clone 245 demonstrated increased levels of PR-1
transcripts as early as 6 h after the initiation of ozone
exposure, with the maximum level (24-fold) being reached after the 2nd
d of fumigation. This high level of PR-1 transcript accumulation was
attained on each of the subsequent days of exposure (Fig. 3B).
Recent work has indicated that in tobacco, ozone protection can
be induced by wounding and treatment with jasmonates before ozone
exposure (Örvar et al., 1997). To examine the role of
JA-mediated gene expression in ozone responses, we measured the levels
of WIN3.7 mRNAs in ozone-treated poplar plants.
WIN3.7 is a wound-inducible gene from poplar that is
homologous to the trypsin family of proteinase inhibitors (Bradshaw et
al., 1989) that can also be induced by exogenous application of MeJA
(Fig. 4). In clone 245, levels of WIN3.7 mRNA were induced up to 20-fold by ozone, whereas in
clone 388, only a 6-fold induction was observed. Similar to the
induction pattern of PAL, WIN3.7 transcript
levels were induced maximally by 3 h, then returned to control
levels by 24 h (Fig. 3A). This induction was evident every day
during the 4 consecutive d of ozone exposure (Fig. 3B).
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| Figure 4.
Induction of WIN3.7 mRNA by MeJA.
Clone 245 plants (6- to 8-week-old cuttings) were sprayed with an
aqueous solution containing either 0.1% Triton X-100 (Control) or
0.1% Triton X-100 containing 100 mM MeJA until the leaves
were completely wetted. At the indicated times after spraying, the
second and fourth fully expanded leaves were harvested. Total RNA was
extracted, subjected to RNA-blot hybridization analysis, and the amount
of hybridizing radioactivity was quantitated using a phosphor imager.
Time points represent the hours after the beginning of the
treatments.
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Wound- and Pathogen-Induced Gene Expression
Comparisons of the ozone-induced patterns of defense-gene
expression in ozone-sensitive and -tolerant clones of hybrid poplar indicated that clone 388 has a greatly attenuated ozone response with
respect to ozone-induced gene expression. To determine if this lack of
defense-gene activation was specific to ozone treatment, we examined
the patterns of gene expression in wounded leaves and in leaves
infiltrated with an avirulent P. syringae pv
maculicola strain. Wounding treatment appeared to induce
PAL, OMT, and WIN3.7 expression in
both clones (Fig. 5). However, the extent
of this induction was far greater in the ozone-tolerant clone 245 than in the ozone-sensitive clone 388. In clone 245, wounding induced PAL mRNA levels 10-fold above unwounded controls, in
contrast to ozone, which induced PAL levels by only 3- to
5-fold above ambient-air-grown controls (Fig. 5). Induction of
PAL transcripts occurred as early as 6 h after
wounding, peaked at 30 h, and remained elevated throughout the
4 d of wounding. In clone 388, wound-induced levels of
PAL mRNA were barely detectable above the control levels. OMT transcript accumulation was also induced by wounding to
a greater extent in clone 245 (Fig. 5). The highest expression level of
OMT mRNA (3- to 5-fold above the controls) was not reached until 54 h after the start of the experiment. Expression of the wound- and MeJA-inducible WIN3.7 gene increased in both
clones (Fig. 5). However, clone 245 showed an increase of
WIN3.7 mRNA up to 15-fold greater than the levels seen in
clone 388 after wounding. PR-1, as expected, was not induced
in either clone 388 or 245 by wounding (data not shown).
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| Figure 5.
Comparison of wound-induced accumulation of
PAL, OMT, and WIN3.7 mRNAs
in hybrid poplar clones 245 and 388. Total RNA was extracted from the
combined second and fourth fully expanded leaves of 6- to 8-week-old
cuttings that had been wounded as described in the text. Total RNA was
extracted, subjected to RNA-blot hybridization analysis, and the amount
of hybridizing radioactivity was quantitated using a phosphor imager.
Time points represent the hours after the initial wounding event.
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To determine if defense-gene expression via a SA-dependent pathway
activated by pathogen infection was also reduced in the ozone-sensitive
clone 388, we used the bacterial phytopathogen P. syringae
pv maculicola KD4326 to induce a nonhost HR and monitored the induction of PR-1 mRNA accumulation. Infiltration of
bacteria into leaves produced visible lesions by 12 h in both
clones. Lesion formation was strictly limited to the site of
infiltration, and the timing of lesion appearance was not significantly
different between the clones. PR-1 transcripts were induced
in both clones but, again, the magnitude of this induction was
significantly less in the ozone-sensitive clone 388 (Fig.
6). At 24 h after infiltration,
PR-1 transcript levels were induced only 2-fold over the
mock-inoculated controls in clone 388 compared with a 10-fold induction
in clone 245.
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| Figure 6.
Comparison of pathogen-induced PR-1
mRNA accumulation in hybrid poplar clones 245 and 388. Leaves of
8-week-old cuttings were infiltrated with either P. syringae pv maculicola KD4326 (Psm) in 10 mM MgCl2 or mock inoculated with 10 mM MgCl2 (Mock). Total RNA was extracted from
the infiltrated area of the second through fifth fully expanded leaves,
subjected to RNA-blot hybridization analysis, and the amount of
hybridizing radioactivity was quantitated using a phosphor imager. Time
points represent the hours after infiltration.
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DISCUSSION |
Ozone is known to induce a variety of stress responses in plants
at both the physiological and molecular levels (for review, see
Kangasjärvi et al., 1994; Iqbal et al., 1996; Sandermann, 1996;
Heath and Taylor, 1997; Sharma and Davis, 1997). However, few studies
have connected ozone-induced physiological responses to the underlying
changes in gene expression, particularly in woody tree species. In our
study, we compared changes in physiological and molecular responses in
two hybrid poplar clones that exhibit different sensitivities to ozone.
We found that specific differences in both physiological changes
and gene-expression patterns observed in the two clones correlate with
their ozone sensitivity.
The phenomenon of ozone causing a decrease in photosynthetic rate and
stomatal conductance has been reported previously in hybrid poplar
(Sen-Gupta et al., 1991; Pell et al., 1992) and other plant species
(Dann and Pell, 1989; Darrall, 1989). We also found that ozone reduced
stomatal conductance and photosynthesis in both of our poplar clones.
However, after 2 d of ozone exposure the percentage reduction in
stomatal conductance was significantly greater in the ozone-tolerant
clone than in the ozone-sensitive clone. Because the stomata regulate
the entry of gaseous pollutants into the plant, they may play an
important role in determining plant sensitivity to ozone (Iqbal et al.,
1996). Although both clones demonstrated an active avoidance response
of stomatal closure during exposure to ozone, the ozone-tolerant clone
responded more quickly. Thus, the tolerant clone may have excluded more
ozone, resulting in less visible damage to the leaves.
After 4 d of ozone exposure, photosynthesis was reduced in both
clones. However, stomatal conductance in ozone-treated plants actually
increased slightly in clone 245 from d 2 to 4, but still remained below
levels measured in ambient-air-treated plants. The percentage reduction
in stomatal conductance in clone 245 decreased from 80% on d 2 to 12%
on d 4. This is accounted for not only by the increase in stomatal
conductance rate in ozone-treated clone 245 plants, but also by a 53%
decrease in stomatal conductance in ambient-air-treated clone 245 plants. This decrease in stomatal conductance in ambient-air-treated
clone 245 is likely related to the change in biomass within the
chambers. Clone 245 is an extremely fast-growing variety and exhibited
considerable growth and significant increases in biomass during the 4-d
experimental period (data not shown). The increase in biomass was not
accompanied by an increase in the rate of watering and may have led to
a shortage of water to the plant, which could cause a decrease in
stomatal conductance (Winner et al., 1988). However, even with the
decrease in stomatal conductance observed in the control clone 245 plants, the ozone-induced reduction was greater.
Differences were also observed in defense-related gene expression
between the ozone-tolerant and ozone-sensitive clones. A transient
5-fold induction of PAL in the ozone-tolerant clone 245 was
observed, which reached a maximum level at 3 h after treatment. These results are consistent with those reported for ozone-treated herbaceous species (Eckey-Kaltenbach et al., 1994; Sharma and Davis,
1994) and a deciduous tree (Tuomainen et al., 1996). However, the level
of ozone-induced PAL transcripts in the ozone-sensitive clone 388 was 4-fold less compared with the tolerant clone. During the
4 d of ozone exposure, the levels of ozone-induced PAL
transcripts in the ozone-sensitive clone remained significantly below
the levels attained in the ozone-tolerant clone. Transcripts for OMT, a
phenylpropanoid-biosynthetic enzyme involved in lignin formation, were
induced 5-fold by ozone in clone 245. This induction was transient,
with maximum expression reached at 12 h after treatment. The
difference in PAL and OMT
transcript-induction kinetics is likely attributable to the fact that
OMT is active downstream in the phenylpropanoid-biosynthetic
pathway from PAL. As was observed with PAL mRNA, induction
of OMT mRNA in clone 388 was only 2-fold above that in
ambient-air-grown controls throughout the 4-d exposure.
The induction of phenylpropanoid biosynthesis by ozone is well
documented; thus, the reported induction of PAL and
OMT transcripts by ozone in hybrid poplar is not surprising.
In addition to the ozone induction of PAL and OMT
transcripts, ozone treatment has been shown to cause increased
isoflavonoid and flavonoid biosynthesis in soybean (Keen and Taylor,
1975). Ozone-induced increases in the activities of phenylpropanoid and
isoprenoid biosynthetic enzymes in pine have also been reported
(Rosemann et al., 1991; Wegener et al., 1997). The observation that the
phenylpropanoid pathway is induced in a variety of plants by ozone
provides correlative evidence that synthesis of phenylpropanoid
derivatives may play a protective role during ozone stress. This
protective effect may be related to the ability of plant phenolics and
flavonoid derivatives to function as antioxidants because of their
ability to trap free radicals (Lewis, 1993). Thus, the increased ozone sensitivity of clone 388 may be attributable in part to the lack of
sufficient induction of the phenylpropanoid pathway to provide protective levels of these antioxidant compounds.
Our results concerning the induction of PAL mRNA in hybrid
poplar differ from those reported by Tuomainen et al. (1996), in which
ozone-induced PAL transcript levels were the same in both ozone-sensitive and ozone-insensitive birch clones. That study also
compared polyamine levels and enzyme activities of superoxide dismutase, peroxidase, and glutathione reductase and found that they
reached higher levels in the ozone-sensitive birch clone compared with
the ozone-insensitive clone. They concluded that higher levels of
putrescine and AOS-scavenging enzymes correlated with the appearance of
physical damage. The differences in PAL induction in
ozone-sensitive and ozone-tolerant hybrid poplar and birch varieties
may be related to different mechanisms of lesion formation in these two
experimental systems. The hypothesis that different mechanisms of ozone
sensitivity are important in different pairs of ozone-sensitive and
ozone-tolerant plants is consistent with the results of Wellburn and
Wellburn (1996). In their study it was found that changes in levels of
polyamines, phenols, reduced glutathione, reduced ascorbate, and total
ascorbate in pairs of tolerant or sensitive selections or cultivars of
six different species were not correlated with either tolerance or sensitivity. Increased ethylene emission was consistently observed in
the more ozone-sensitive plants.
Previous work in this laboratory has demonstrated that ozone activates
at least two distinct signaling pathways in Arabidopsis, one of which
overlaps with the HR and SAR activation pathways and is SA dependent
(Sharma et al., 1996). To examine the potential role of this pathway in
hybrid poplar, we used PR-1 as a marker gene. In clone 245, PR-1 was induced 24-fold over ambient-air-grown controls,
reaching maximum levels 12 h after the start of ozone treatment.
This result is consistent with the ozone-induced accumulation of the
PR-1 transcripts in Arabidopsis reported by Sharma et al. (1996). Again, a difference between the ozone-tolerant and
ozone-sensitive clones was observed. Induction of PR-1 in
clone 388 reached levels only 2- to 3-fold over those of
ambient-air-grown controls, about 10-fold less than the levels attained
in the tolerant clone. Because activation of a SA-dependent pathway may
provide some protection to ozone (Sharma et al., 1996), the apparent
lack of a significant induction of SA-dependent gene expression may
have enhanced the ozone sensitivity of clone 388. A significant
difference in the induction of PR-1 mRNA accumulation was
also observed in clones 245 and 388 in response to infection with an
avirulent bacterial pathogen; PR-1 transcript levels were
20-fold higher in the ozone-tolerant clone 245 than in clone 388 (Fig.
6). These results demonstrate that attenuated PR-1
expression is not specific to ozone exposure and suggests that clone
388 may have generally diminished SA-mediated stress responses.
The overlap in the induction of SA-mediated responses by both ozone and
pathogens is likely because both stresses result in the production of
AOS. Increased production of AOS by ozone upon entering the plant could
mimic the "oxidative burst," which in plant-pathogen interactions
is thought to trigger the signaling events that activate the HR and
SAR, resulting in disease resistance (for review, see Mehdy et al.,
1996; Lamb and Dixon, 1997). It has been proposed (Sharma and Davis,
1994; Sharma et al., 1996) that the necrotic lesions observed in some
ozone-treated plants may be caused by the activation of the programmed
cell death component of the HR (for review, see Dangl et al., 1996;
Greenberg, 1997). Our results on PR-1 induction indicate
that the ozone-sensitive hybrid poplar clone 388 has an attenuated
SA-mediated response that would normally lead to the formation of these
ozone-induced HR-like lesions. Because the sensitive clone 388 develops
much larger regions of necrosis and the tolerant clone 245 develops smaller, HR-like lesions, it could be argued that these lesions develop
via two distinct mechanisms. In the sensitive clone 388, the
ozone-induced lesions may be caused by the toxic effects of AOS that
accumulate because of the lack of any induced defense responses. In the
ozone-tolerant clone 245, ozone may induce lesions by activating a
cell-death pathway associated with HR. This interpretation is
consistent with previous studies in tobacco, in which the
ozone-sensitive cv Bel-W3 exhibited higher levels of PR gene expression
compared with the more tolerant cv Bel-B (Ernst et al., 1992;
Schraudner et al., 1992). Similar results were obtained in comparisons
of tolerant and sensitive ecotypes of Arabidopsis (Sharma and Davis, 1997; I. Aguilar, Y. Sharma, and K. Davis, unpublished data). In cv
Bel-W3 and the highly ozone-sensitive Arabidopsis ecotype, the apparent
increased ozone sensitivity may be attributable to an enhanced HR in
response to the ozone-induced production of AOS.
Evidence that a second, SA-independent pathway is also activated by
ozone, resulting in the induction of PAL transcripts in Arabidopsis, was presented by Sharma et al. (1996). The possibility that JA may be involved in ozone-activated signal transduction pathways
was addressed by Örvar et al. (1997), who demonstrated that pretreatment of tobacco by either mechanical wounding or JA
decreased the amount of ozone injury in tobacco plants. We confirmed
that the wound-induced WIN3.7 gene is induced by MeJA and
used this gene as a marker for JA-mediated responses. We found that
ozone induced WIN3.7 transcript accumulation in the
ozone-tolerant clone 245 by 20-fold over ambient-air-grown controls.
This same transcript was only induced 3-fold in the ozone-sensitive
clone 388. The induction of WIN3.7 transcripts cannot be an
indirect response to ozone-induced lesion formation because transcript accumulation was detected at 3 h after the start of ozone
treatment, whereas lesions in clone 245 were not visible until between
12 and 24 h. Furthermore, if induction of this gene is a
by-product of lesion formation, it would be expected to correlate with
the level of injury development. However, in clone 388, which develops the most severe lesions, WIN3.7 transcript levels were less
than 10% of the levels reached in clone 245.
To determine if the reduced levels of defense-gene expression in clone
388 were caused only by the inability to respond to ozone stress, we
performed wounding experiments. Wounding induced transcript levels of
PAL, OMT, and WIN3.7 in both the
sensitive and tolerant clones. However, wound induction of these
transcripts in clone 388 was significantly reduced compared with that
in clone 245. Thus, the ozone-sensitive clone appears to have greatly
attenuated ozone- and wound-induced responses, indicating that these
responses may share at least a portion of the same signaling pathway. A potential link between ozone- and wound-induced responses is JA. JA is
associated with a variety of physiological responses (for review, see
Sembdner and Parthier, 1993; Reinbothe et al., 1994; Creelman and
Mullet, 1997), many of which correlate with ozone responses. For
example, exogenous application of JA has been reported to accelerate
leaf senescence, promote stomatal closure, inhibit photosynthetic
activity, inhibit Rubisco biosynthesis, and induce defense-gene
expression.
We found that clone 245, upon exposure to ozone, displays inhibition of
photosynthetic activity, enhanced stomatal closure, and induction of
both PAL and a proteinase- inhibitor gene. Furthermore, studies by Landry and Pell (1993) on this same clone indicated that
ozone caused accelerated leaf senescence and inhibited both Rubisco
activity and photosynthesis. The ozone-sensitive clone 388 also
exhibits reduced photosynthetic activity and stomatal conductance,
confirming results by Eckardt et al. (1991) and Pell et al. (1992).
However, the data reported by Landry and Pell (1993), Eckardt et al.
(1991), and Pell et al. (1992) did not include direct comparisons
between these two hybrid poplar clones. Our data include measurements
of photosynthetic rate and stomatal conductance that were performed
under identical conditions for both clones so that the responses may be
compared. These results indicate that the ozone-induced reduction in
stomatal conductance in clone 388 plants is significantly (P < 0.05) less than the reduction measured in clone 245 plants. The
ozone-induced reduction in photosynthetic rate did not differ
significantly between the clones, which may be attributable to high
variability between individuals combined with a small sample size. In
addition, when photosynthetic rates were measured under saturating
light, the ozone-induced reduction in photosynthetic rate in clone 245 plants was significantly greater than the reduction measured in clone 388 plants (data not shown). This may indicate that the greater reduction in photosynthetic rate observed in clone 388 compared with
clone 245, although not statistically significant, may have biological
significance. Furthermore, we observed very little senescence
attributable to ozone treatment (data not shown) and a reduced level of
proteinase-inhibitor gene expression in clone 388 compared with clone
245. All of these results are consistent with JA having a role in
mediating some ozone-induced responses in hybrid poplar.
The apparent attenuated JA-mediated responses observed in clone 388 may
act as an important component of its increased ozone sensitivity by
causing a slower rate of stomatal closure. Thus, in clone 388, it is
possible that more ozone enters the mesophyll and generates more AOS,
resulting in more tissue necrosis. The effects of increased ozone
entering the mesophyll may be compounded by the lack of SA-dependent
inducible antioxidant defense responses. Alternatively, the enhanced
ozone sensitivity of clone 388 may not be the direct result of the
reduced levels of defense-response induction but, rather, may be caused
by the increased stomatal conductance and higher levels of AOS
production that exceed the antioxidant capacity of the cells. This
could result in sufficient cell damage to prevent the defense responses
from being activated. This alternative seems less likely given the
observation that the timing of lesion development was very similar in
clones 388 and 245 and occurred well after early defense-gene
activation was observed. It would be expected that more rapid and
extensive cell damage would be associated with more rapid necrosis.
Moreover, the defense responses of clone 388 were attenuated not only
in response to ozone, but also in response to wounding and pathogen treatments. Thus, it is more likely that the increased ozone
sensitivity of clone 388 is related to the reduced activation of
defense signaling pathway(s) that are common for all three stresses,
e.g. a reduced ability to generate or respond to stress signals such as
ethylene, JA, and SA.
Further study of the differential ozone sensitivity of the hybrid
poplar clones 245 and 388 should prove important in characterizing further the mechanisms of interaction between ethylene-, SA-, and
JA-activated defense-response pathways during exposure to oxidative
stress. The current study will allow a comparison of the ozone-induced
responses in a woody tree species with the responses of herbaceous
plants such as tobacco and Arabidopsis. In addition, this system will
be a valuable tool in defining novel signaling pathways for
defense-gene induction in trees and for the identification of specific
genes that may be useful in increasing stress resistance.
| |
FOOTNOTES |
1
This work was supported in part by funds
provided by the U.S. Department of Agriculture, Forest Service,
Northeastern Forest Experiment Station.
*
Corresponding author; e-mail davis.68{at}osu.edu; fax
1-614-292-5379.
Received May 20, 1998;
accepted August 31, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AOS, active oxygen species.
HR, hypersensitive
response.
JA, jasmonic acid.
MeJA, methyl jasmonate, OMT,
O-methyltransferase.
PAL, Phe ammonia-lyase.
PR, pathogenesis-related.
SA, salicylic acid.
SAR, systemic acquired
resistance.
| |
ACKNOWLEDGMENTS |
We thank Eva Pell (The Pennsylvania State University, University
Park) for supplying the initial cuttings of clones 245 and 388 and for
her expertise and advice. We also thank Carl Douglas, Marc Van
Montagu, and Milton Gordon for supplying the cDNA probes used in this
study, and Staci Putney (The Ohio State University) for maintaining the
hybrid poplar plants.
| |
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Abstract
Introduction